The viruses of the TBEV complex cause human disease of varying severity with up to 30% mortality. Formalin-inactivated TBEV vaccines are available in Europe, they are not licensed in the US and do not afford lifetime protection. A live attenuated TBEV vaccine is expected to induce a higher level and more durable immunity than that induced by an inactivated virus vaccine. Three antigenic chimeric TBEV vaccine candidates were generated and compared for level of replication in neuroblastoma cells, for virulence in mice, and for safety, immunogenicity and efficacy in rhesus monkeys. Two chimeras were generated by replacing the membrane and envelope protein genes of dengue-4 virus (DEN4) with the corresponding genes of a Far Eastern TBEV in the presence (TBEV/DEN4d30) or absence (TBEV/DEN4) of a 30 nucleotide deletion (d30) in the 3? UTR of the genome. A third vaccine candidate was based on the antigenically related, naturally attenuated Langat virus (LGT). Chimerization of LGT with DEN4 resulted in: (i) decreased neurovirulence and neuroinvasiveness in mice; (ii) highly restricted viremia in monkeys; (iii) highly restricted replication in cells of neural origin. In contrast, TBEV/DEN4 and TBEV/DEN4d30 were neither attenuated for neurovirulence in suckling mice nor restricted in replication in the neuroblastoma cells. However, both were attenuated for neuroinvasiveness in mice. TBEV/DEN4 replicated to moderately high titer in monkeys (mean peak viremia = 1260 PFU/ml) indicating that the TBEV/DEN4 chimerization had only a modest, if any, attenuating effect in monkeys. The addition of the d30 mutation to TBEV/DEN4 greatly attenuated the chimeric virus for monkeys (mean peak viremia = 5 PFU/ml) and induced a higher level of neutralizing (NT) antibody against TBEV than did LGT/DEN4. A single dose of either TBEV/DEN4d30 or LGT/DEN4 or three doses of an inactivated TBEV vaccine protected monkeys against LGT challenge. Clinical evaluation of LGT/DEN4 vaccine in humans was initiated at Vanderbilt University Medical Center. [unreadable] Prior to clinical evaluation of the TBEV/DEN4d30 vaccine candidate in humans, it was necessary to further examine its neurovirulence in mice and monkeys. Since a naturally attenuated LGT is the only member of the TBEV complex that was previously tested in humans as a live vaccine, TBEV/DEN4d30 and LGT viruses were compared in adult mice for their abilities to replicate and cause CNS disease following intracerebral (IC) inoculation. LGT replicates efficiently in the brain of mice, attains a peak titer of 9.6log10 PFU/g, and causes encephalitis or death on day 7. However, TBEV/DEN4d30 attained a peak virus titer of only 1.9log10 PFU/g, indicating that TBEV/DEN4d30 was remarkably attenuated compared to LGT vaccine virus. A comparative study of the pathology produced by the TBEV/DEN4d30 and LGT viruses in monkeys inoculated IC was recently initiated. If TBEV/DEN4d30 will be satisfactorily attenuated, a clinical lot will be prepared for evaluation in humans. If this study indicates that additional attenuation is needed, this might be achieved by introducing one or more attenuation mutations identified for the LGT virus. In our effort to identify attenuating point mutations, a panel of 5-fluorouracil LGT mutants was screened for temperature sensitivity and decreased replication in neuroblastoma cells. One of these ts mutants, clone E5-104, exhibited a >1000-fold reduction in replication in neuroblastoma cells and lacked detectable neuroinvasiveness for immunodeficient mice. Using reverse genetics, we demonstrated that the Lys46 > Glu substitution in NS3 as well as the Lys315 > Glu change in E were responsible for the observed attenuation phenotypes and provided a useful foundation for further development of a TBEV vaccine. Evaluation of the sequence of virus recovered from brain of SCID mice inoculated with LGT mutants identified sites in the LGT genome that promoted neurovirulence/neuroinvasiveness and might be useful in further understanding the molecular basis of the pathogenesis of neurotropic flaviviruses.[unreadable] [unreadable] Vaccines against West Nile virus (WN) and St. Louis encephalitis virus (SLE).[unreadable] [unreadable] WN illness is an emerging disease in the USA and represents a significant public health threat. A live attenuated virus vaccine is being developed in the LID to protect humans against WN disease. Previously, we found that chimeric viruses (WN/DEN4 and WN/DEN4d30) bearing the structural protein genes of wild-type WN were attenuated and efficacious in mice and monkeys against WN challenge. We have reported the generation of a clinical lot of WN/DEN4d30 virus and its preclinical evaluation for safety and immunogenicity in mice, geese, and monkeys. Unlike its WN parent, the vaccine candidate was not neuroinvasive in SCID mice, had greatly reduced neurovirulence in suckling mice, and replicated poorly in neuroblastoma cells as well as in brains of suckling mice. WN/DEN4d30 failed to infect geese, indicating that the vaccine virus would be restricted in its ability to be transmitted from vaccinees to domestic or wild birds. In monkeys, the WN/DEN4d30 vaccine was highly immunogenic despite its low level of replication with undetectable viremia. Furthermore, the WN/DEN4d30 vaccine was safe and readily induced WN NT antibodies even in monkeys immune to each of the four serotypes of dengue virus. These studies indicate that this vaccine should be efficacious for the recipient and safe for the environment. Currently, a Phase I clinical trial in humans is underway with the WN/DEN4d30 vaccine at the Johns Hopkins School of Public Health. [unreadable] Before the 1999 outbreak of WN, SLE had been the dominant flavivirus causing encephalitis in the USA. SLE vaccine development was initiated using the chimeric approach found successful for WN and TBEV. Chimeric SLE/DEN4 and SLE/DEN4d30 viruses that contained the structural protein genes of SLE (strain Hubbard) were recently generated. These viruses were highly attenuated in mice, i.e., they exhibited reduced neurovirulence in suckling mice and lacked neuroinvasiveness in adult mice. Efforts are underway to further evaluate the level of attenuation and immunogenicity of these viruses in mice and monkeys.